Most recent version of protocol: 2/14/96
Dr. Paul J. Zambino, Research Plant Molecular Pathologist,
U.S.D.A. Forest Service, North Central Forest Experiment Station,
Forestry Sciences Laboratory, 5985 Hwy. K, Rhinelander, WI, 54501.
Phone: (715)362-1178. EMAIL: paulz@puccini.crl.umn.edu.
Overview of method: This protocol modifies mini-pestle grinding techniques of Doudrick et al., (1993) by grinding Cronartium spores desiccated to low relative humidity in the absence of buffer. This protocol neither requires spores to be lyophilized before grinding, nor liquid nitrogen or dry ice to be used during grinding as in other methods (Hamelin et al., 1995; Sun et al., 1995). Desiccated spores are mixed with diatomaceous earth in microfuge tubes, frozen before use, then kept cold over ice. Spores are ground using plastic mini-pestles mounted in an electric drill.
DNA obtained using this protocol does not become sheared as in protocols that grind spores in liquid, perhaps because the DNA is not released from cracked spores until after the liquid is added. After grinding, DNA is extracted from the cracked spores by incubation in an SDS extraction buffer at 65 C, and the crude extract is purified by organic extractions, ethanol precipitation, RNAse treatment, two extractions over Strataclean resin (Stratagene), and a second ethanol precipitation. DNA yields from 50 ul spore samples average 2.0 ug, and display a uniform band of fragment size > 23kb after electrophoresis.
Recipe to make 50 ml of SDS BME lysis buffer, 1X concentration:
Aliquot as needed into small tubes that can be tightly capped. I prefer sterile plastic culture tubes that hold 3.5 ml buffer. Add 35 ul (=1 % v/v) b-mercaptoethanol (BME) to individual tubes just before extractions.
Preliminary: Desiccate aeciospores or urediniospores by storing at either 0 or 30 RH at 4C for up to a week over either a dry-type desiccant or a solution of 142 g KOH/200ml water, respectively (this desiccation step is equivalent to that used prior to viable spore storage at - 80C). Maintain a supply of disposable culture tubes containing frozen 1.7 ml aliquots of TRIS-equilibrated phenol, and check if there are sufficient tubes for the planned extractions (1 tube per 6 extractions). Cut 2.5 cm wide strips of parafilm or alternative stretch wrap for keeping the chuck of the drill clean, 1 strip per extraction.
1) The day before: Spore preparation. Place ca. 50 ul (10 - 75 ul) of desiccated aeciospores or urediniospores into 1.5 ml centrifuge tube. Use of a biological safety hood is optional but is recommended while measuring and grinding spores. Estimate approximate spore volume by compacting spores into the end of the tube by tapping the bottom of the tube onto the bench, then comparing the level of compacted spores to that of 50 ul water in a reference tube and adjusting volume as needed. For desiccated aeciospores or urediniospores, 50 ul is roughly equivalent to 25 ug. To each sample of desiccated spores, add 50 ul (an equal volume) acid washed diatomaceous earth (DE) that has been autoclaved, thoroughly dried in an oven, and measured into 50 ul quantities as described for spores. Spores and DE are thoroughly mixed with a sterile dissecting needle and spores kept frozen at -80 C until extraction.
2) Day of extraction, Preliminary: Set hot blocks and/or water baths to 65 C, 55 C, 37 C and turn on speed vacuum to pre-cool its moisture trap. For every 6 extractions, mix a disposable plastic culture tube of 3.4 ml of Doudrick lysis buffer with 34 ul BME and place in hood, and thaw 1 tube of 1.7 ml TRIS equilibrated phenol in hood. Put 6 spore samples on ice. Wipe down area, drill, pipettes, etc. with ethanol.
3) Grinding method: In hood, mount plastic pestle in chuck of variable speed electric drill used only for DNA extraction. Wrap the drill chuck and the base of the pestle with parafilm. Stir sample briefly using a sterile dissecting needle and set needle aside. Place the tube into a small beaker half full of wet ice, holding the tube firmly against the bottom and side walls of the beaker. To grind sample, gently lower pestle to the bottom of the spore mix, then begin to grind spores at low to medium speed. Allow the pestle to firmly contact the tube bottom 2 times every second, slightly changing the initial angle of the pestle between strokes. Every 30 strokes (15 sec), use the needle to stir the spore mix and dislodge spores that have become packed into the bottom of the tube. Grind sample for 4 cycles of 30 strokes, more if necessary for different rust species.
4) Extraction: Add 525 ul Doudrick's SDS BME buffer to the tube, stirring buffer and spores with needle. Vortex briefly until spores are dispersed, then put tube on ice. Change pestle, parafilm, and dissecting needle after each sample, and wipe several times with ethanol all equipment, surfaces, gloves, etc. that spores may have contacted. Resuspend material that has settled in the tubes after all six samples have been processed.
5) Heat treatment: Incubate tubes in 65 C hot block for 1 hr. Flick tubes to mix every 10 - 15 min. during incubation. Prepare phenol:chloroform by adding 1.7 ml 15:1 Chloroform:isoamyl alcohol (SEVAG) to thawed tubes of 1.7 ml TRIS equilibrated phenol, mix, and allow to settle. If 12 samples are being processed, grind second set of samples during heat treatment of the first 6. If only one hot block is available, set hot block to 55 C for RNAse treatment as soon as last samples are removed from 65 C.
6) Organic extractions: For each organic extraction, form emulsion by inverting tubes ca. 20 times, spin at 10,000 g for 15 min., and place supernatant in new tubes. Extraction 1: use 525 uL phenol:chloroform, recover 450 ul supernatant. Extraction 2: use 450 ul SEVAG, recover 400 ul supernatant.
7) ETOH precipitation: Bring volume up to 400 ul if needed using TE-8 (10 mM TRIS, 0.1 mM EDTA, pH 8.0). Add 40 ul (0.1 vol.) 3M Sodium acetate pH 5.3 and mix by inversion. Add 880 ul cold (-20 C) absolute ETOH and mix by inversion. Chill on ice or - 20 C 30 min. Centrifuge at top speed 5 min. Pour off supernatant. Rinse pellet in 1000 ul cold (-20 C) 70% ethanol, inverting tube several times. Centrifuge at top speed 5 min., pour off, centrifuge a few seconds, pull off remaining supernatant with a pipette tip. Dry pellet 30 min. in speed vac.
8) Resuspend pellet in 100 ul TE-8; heat to 55 C 10 min. to ensure complete disassociation of pellet.
9) RNAse treatment: Add 5 ul of 10mg/ml RNAse. Incubate 15 min. at 55 C, 15 min. at 37 C.
10) Strataclean purification, 2 times: Add 14 ul fully suspended Strataclean resin slurry, mix 2 min. by flicking, centrifuge 2 min., transfer 100 ul supernatant to fresh tube, being careful not to carry over any of the resin on the last extraction.
11) Final selective ETOH precipitation: To 100 ul DNA solution add 43 ul 5 M NaCl (0.427 volumes) to bring to 1.5 M NaCl concentration. Add 286 ul chilled absolute ETOH (2 volumes), hold on ice or -20 C 30 min. Centrifuge at top speed for 5 min., rinse in 70 % ETOH as previously described, dry in speed vac and resuspend pellet in 100 ul TE-8.
12) Quantification and storage: Use 2.0 ul DNA solution + 8 ul TE-8+ 2 ul dye for gel electrophoresis for initial examination of DNA concentration and quality. Use a DNA standard that has bands in the 40-60 ng range. Freeze DNA stocks at -20 C for later use.
Optional stopping spots during extraction: During ethanol precipitation, or after pellet drying. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.
References for additional DNA protocols:
Doudrick, R. L., W. L. Nance, C. D. Nelson, G. A. Snow, and R. C. Hamelin. 1993. Detection of DNA polymorphisms in a single urediniospore derived culture of Cronartium quercum f.sp. fusiforme. Phytopathology 83:388-392.
Hamelin, R. C., J. Beaulieu, and A. Plourde. 1995. Genetic diversity in populations of Cronartium ribicola in plantations and natural stands of Pinus strobus. Theor. Appl. Genet. 91:1214-1221.
Sun, L.-J., J. E. Carlson, and B. J. van der Kamp. 1995. A rapid procedure for preparing high molecular weight DNA from rust aeciospores for RAPD analysis. Proc. 4th IUFRO Rusts of Pines Working Party Conf., Tsukuba: 143-148.
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